The present invention relates to a rapid and readily reproducible process for stabilizing biologically active substances by combining the biologically active substance with a stabilizing mixture and drying the resulting mixture into a dry, amorphous product by means of convection drying. The invention also relates to the amorphous, microscopically homogeneous products which are obtained by this process, are in the form of powders and have a uniform geometric, in particular spherical, shape. The invention furthermore relates to the use of substance mixtures for stabilizing biologically active material, in particular proteins by means of spray drying.
It is well known that proteins, especially human proteins, can be stabilized in solid preparations by a large number of substances, preferably by sugars or combinations of sugars and amino acids.
Various processes and formulations for producing dry, biologically or therapeutically active materials are described. By dry materials are meant substances and substance mixtures which have a residual moisture content not exceeding 8% (g/g), preferably not exceeding 4% (g/g), particularly preferably not exceeding 2%. Freeze-drying processes are widely used for producing such formulations [F. Franks, Cryo Lett. 11, 93-110, (1990); M. J. Pikal, Biopharm. 3 (9), 26-30 (1990); M. Ilora, Pharm. Research 8 (3), 285-291 (1992); F. Franks, Jap. J. Freezing Drying 38, 15-16, (1992)] but have the specific disadvantages of freeze-drying. They consume large amounts of energy, require the use of refrigerants, some of which are environmentally harmful (frigens), and are time-consuming because it is often necessary to remove relatively large volumes of ice by sublimation. The freezing step necessary for freeze-drying may be destabilizing for a large number of substances, especially for proteins. This process is therefore not applicable at all to some biological materials.
Alternatives to freeze drying for producing dry protein preparations are processes which dry the material by using heat and/or vacuum [F. Franks, R. M. H. Hatley: Stability and Stabilization of Enzymes; Eds. W. J. J. von den Teel, A. Harder, R. M. Butlaar, Elsevier Sci. Publ. 1993, pp. 45-54; B. Roser, Biopharm, 4 (9), 47-53 (1991); J. F. Carpenter, J. H. Crowe, Cryobiol. 25, 459-470 (1988)]. Examples which may be mentioned in this connection are vacuum drying with or without use of elevated temperature, spray-drying processes in a wide variety of modifications, including combined use of vacuum and spraying techniques, and drum drying and other thin-film drying processes.
J. F. Carpenter, J. H. Crowe, Biochemistry 28, 3916-3922 (1989); K. Tanaka, T. Taluda, K. Miyajima, Chem. Pharm. Bull. 39 (5), 10-94 (1991), DE 35 20 228, EP 0 229 810, WO 91/18091, EP 0 383 569 and U.S. Pat No. 5,290,765 describe preparations which contain sugars or sugar-like substances. However, the production of dry, carbohydrate-containing preparations by means of freeze or vacuum drying, especially of sugar preparations, is associated with disadvantages in the state of the art. These include, inter alia, high energy consumption for drying to an acceptable residual moisture content, extended process times at low drying temperatures, formation of highly viscous, water-containing masses (called xe2x80x9crubberxe2x80x9d) or glassy melts whose glass transition temperatures are below room temperature. The disadvantages described above significantly influence the stability of biological materials in such preparations.
It is also evident from the literature cited above that preparations suitable for stabilizing proteins should have glassy, that is to say amorphous, structures whose glass transition temperature is above the intended storage temperature. The glass transition temperature (Tg) is the temperature at which an amorphous or partially crystalline solid is converted from the glassy state into the fluid or viscous state and vice versa. This involves drastic changes in the viscosity and, associated therewith, in the diffusion coefficient and the kinetic mobility of the biological materials. Physical characteristics such as hardness and modulus of elasticity are changed, as are the thermodynamic functions of volume, enthalpy and entropy. The glass transition temperature of, for example, a sugar-containing composition and its residual water content are physically linked together in such a way that increasing residual amounts of water lead to declining glass transition temperatures and vice versa. It is thus possible to infer from measurement of the glass transition temperature, for example by differential scanning calorimetry (DSC), whether a preparation has a residual water content suitable for stabilization or, as stated above, a drying process is successful or not. Besides determining the glass transition temperature by means of DSC, the presence of amorphous structures can also be proved by means of X-ray diffraction investigations., optical and electron microscopic examinations.
It was therefore desirable to provide fully amorphous ancillary substance structures for biological or pharmaceutically active materials so that the embedded biological materials can also be kept stable at room temperature and over a long period. The ancillary substance structures should have a low residual moisture content which can be adjusted intentionally, and have a glass transition temperature which is as high as possible.
WO 97/15288 describes a process for stabilizing biological materials by means of drying processes without freezing, with which partially amorphous ancillary substance structures are obtained. The drying was carried out as vacuum drying (at slightly elevated temperatures  less than 50xc2x0 C.), although inhomogeneous products are obtained.
WO 96/32096 describes the production of a homogeneous, dispersible powder, which contains a human protein, carbohydrates and/or amino acids and other ancillary substances, for inhalation by means of spray drying. However, it has emerged that amorphous products are not obtained in any of the examples.
EP 0 682 944 Al describes lyophilisates with pharmaceutically acceptable ancillary substances, consisting of a phase with the protein in amorphous mannitol and a second phase with crystalline alanine. However, these formulations cannot stabilize the biological materials sufficiently well over a lengthy period.
The present invention is based on the object of developing a mild, flexible, readily reproducible, rapid and economic drying process for embedding biological materials, especially human proteins, and of finding stabilizing matrices suitable for this process. It is intended that it be possible with this process to produce fully amorphous and homogeneous products which allow stabilization of the biological materials over a long period.
The present invention relates to a rapid and readily reproducible process for stabilizing biologically active substances by combining the biologically active substance with a stabilizing mixture and drying the resulting mixture into a dry, amorphous product by means of convection drying. The invention also relates to the amorphous, microscopically homogeneous products which are obtained by this process, are in the form of powders and have a uniform geometric, in particular spherical, shape. The invention furthermore relates to the use of substance mixtures for stabilizing biologically active material, in particular proteins by means of spray drying.
The object of the invention, of providing an efficient process for producing amorphous preparations which contain biological materials, is achieved by means of convection drying, in particular by means of spray drying, wherein the air inlet temperature is from about 50 C. to about 300 C. and with the mixtures of at least one zwitterion with polar or apolar radical or a mixture of at least one zwitterion with polar or apolar radical and a carbohydrate. The aforementioned mixtures may also contain ancillary substances such as buffers, surfactants, antioxidants, isotonicizing agents, and preservatives. The invention relates in particular to a process for producing dry, amorphous products which, besides biological material, in particular therapeutically active material, contain substance mixtures for stabilization, which is characterized in that a solution or suspension of biological material and a substance mixture consisting of (a) a carbohydrate and at least one zwitterion with polar or apolar radical or derivatives thereof, and/or (b) at least two zwitterions with polar or apolar radicals or derivatives thereof, and/or (c) at least one zwitterion with polar or apolar radical or a plurality of zwitterions with polar or apolar radicals or derivatives thereof, is produced and dried by means of convection drying with adjustment of a relative moisture content of  less than 70%, preferably  less than 40%, in particular  less than 20%, in the stationary phase at an inlet air temperature of 50-300xc2x0 C., preferably  less than 200xc2x0 C. This manner of drying proves to be particularly advantageous in relation to the stability of the biological materials in the formulations and ensures yields  greater than 90%.
The zwitterions with polar or apolar radicals which are preferably employed are amino carboxylic acids. The substance mixtures employed of group (a) comprise, preferably, mono-, oligo-, polysaccharides, arginine, aspartic acid, citrulline, glutamic acid, histidine, lysine, acetylphenylalanine ethyl ester, alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and/or derivatives thereof. The substance mixtures of group (b) comprise, preferably, arginine, aspartic acid, citrulline, glutamic acid, histidine, lysine, acetylphenylalanine ethyl ester, alanine, cysteine, glycine, isoleucine, leucine, mehionine, phenylalanine, tryptophan, valine and/or derivatives thereof. The zwitter of group (c) are preferably employed in the form of their salts. Preferably used are the salts of arginine, aspartic acid, citrulline, glutamic acid, histidine, lysine, acetylphenylalanine ethyl ester, alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and/or derivatives thereof.
Biologically active materials for the purpose of the invention are one or more substances of the groups of proteins, peptides, glycoproteins, lipoproteins, enzymes, coenzymes, antibodies, antibody fragments, virus constituents, cells and cell constituents, vaccines, DNA, RNA, biological therapeutic and diagnostic agents or derivatives thereof.
It is possible where appropriate to add to the solution or suspension of biological material and the substance mixture of groups (a) and/or (b) and/or (c) conventional ancillary substances from the groups of buffers, surfactants, antioxidants, isotonicizing agents, preservatives.
It is possible by spray drying with the features stated herein to modify carbohydrates, amino acids or derivatives thereof which can be dried with difficulty in such a way that they can be dried, or to mix carbohydrates, amino acids or derivatives thereof with a substance or a substance mixture which increase the Tg so that drying becomes possible and the resulting ancillary substance structure is amorphous and is outstandingly suitable for embedding biological materials. Vacuum drying of comparable formulas results in lower glass transition temperatures.
It has been found that in the case of carbohydrates, especially sugars such as, for example, sucrose, fructose, which indeed form amorphous structures after spraying from aqueous or organic solution into a hot air bed, but have low glass transition temperatures ( less than 20xc2x0 C.) and therefore result in yields which are extremely economically unfavourable on drying, and have low stability on storage, the latter in particular in relation to the retention of amorphous structures, the Tg is increased so much (xe2x89xa720xc2x0 C.) by addition of zwitterions or derivatives thereof that they can be dried in good yield, and the amorphous structure of the carbohydrates or of the complete formulation is retained or stabilized.
It has furthermore emerged, surprisingly, that it is possible to obtain an amorphous structure as is necessary for stabilizing biological materials by using carbohydrates such as, for example, mannitol, after the spraying from aqueous or organic solution into a hot air bed, by adding zwitterions or derivatives thereof prior to spray drying. At the same time, good yields are achieved therein. This result is surprising since no amorphous structures are obtained when the process is carried out without addition of zwitterions.
On the other hand, it has been found that even zwitterions or derivatives thereof which, although they can be dried after spraying from aqueous or organic solution into a hot air bed, are not obtained in amorphous form from this, are obtained in a fully amorphous form on addition of carbohydrates or derivatives thereof.
Amorphous structures are also obtained when zwitterions or derivatives thereof which, although they can be dried after spraying from aqueous or organic solution into a hot air bed, are not obtained in a fully amorphous form from this, can be converted by suitable admixtures of one or more zwitterions into a fully amorphous form. To produce the amorphous structure it is unnecessary to choose only combinations of zwitterions with polar radicals and apolar radicals; on the contrary it is also possible to employ combinations of zwitterions with only polar or only apolar radicals.
It has further emerged that one or more zwitterions with polar or apolar radicals or derivatives thereof which, although they can be spray-dried, are not obtained in amorphous form, can be converted by specific adjustment of the pH of the solution or suspension before the convection drying into the amorphous state, preferably by adjusting to a pH of 2.0-8.0, preferable 7.0-7.5. A specific adjustment of the pH of the solution before the spray drying can also be worthwhile in those cases (variants a and b) where, although amorphous structures are obtained, the stability of the protein in the amorphous structure is to be further improved or the amorphous structure of the sprayed products is to be further increased. Such an adjustment of the pH may also be necessary for physiological reasons.
The skilled person is able, by suitable combination of the variants mentioned, to increase significantly further the effects already achieved. The teaching of the patent allows him to select a zwitterion with polar or apolar radical so that the dried substance mixture has a raised glass transition temperature and/or an amorphous structure by comparison with a substance mixture without a corresponding addition.
The products obtained with the process according to the invention are amorphous and microscopically homogeneous powders in a particle size range from 0.0005 mm to 1 mm, preferably from 0.001 mm to 0.1 mm. The process according to the invention allows preferably spherical particles to be obtained in a particle size range which is adjustable and can be reproduced surprisingly well. The resulting products have a glass transition temperature=20xc2x0 C., preferably 40xc2x0 C., and a residual moisture content  less than 8% (g/g), preferably  less than 4% (g/g). The amorphous structure is furthermore retained over storage times of at least 12 months. Compared with lyophilisates, the products have an apparent density which is higher by at least a factor of 1.15 (15%) than lyophilisates and, compared with products from freeze or vacuum drying, have significantly lower crystalline contents for the same formulation.
The amorphous products, which preferably contain proteins as biological materials, are produced according to the invention by convection drying, in particular by spray drying or spray granulation, by producing a solution or suspension of the biological material and the substance mixture, and carrying out the convection drying at an inlet air temperature of 50-300xc2x0 C., preferably  less than 200xc2x0 C., it having emerged that the relative moisture content in the stationary phase must be adjusted to  less than 70%, preferably  less than 40%, in order to obtain a defined product moisture content of  less than 4%. The process according to the invention thus permits the required residual moisture content in the final products to be adjusted intentionally.
It is possible according to the invention for the convection drying to take place by means of fluidized drying, lift air drying or flight drying. Spray or fluidized bed drying is particularly preferably used according to the invention.
In the spray drying, the material to be dried is sprayed in a manner known per se as solution or suspension through nozzles or by means of a rapidly rotating atomizer disc to give a mist of droplets at the upper end of a wide cylindrical container. The resulting mist of droplets is mixed with hot air or an inert gas, which are passed around the atomizing zone into the dryer. If a solution is atomized under otherwise identical conditions by means of a two-component nozzle or disc, although the particle size distribution of the spray embedding is narrower with disc atomization than with nozzle atomization, it is shifted into the coarser region.
Suitable atomizing devices are swirl pressure nozzles, pneumatic nozzles (two-component/three-component nozzles) or centrifugal atomizers. Although pneumatic nozzles require the greatest energy consumption for atomization per kg of liquid, these nozzles are particularly suitable because of their flexibility, for example for achieving particular particle size ranges or particular particle shapes. The process can also be carried out by means of the use of a three-component nozzle technique. It is possible thereby to atomize two liquids simultaneously.
This makes two variants of the atomization possible:
The two liquids are fed separately and, shortly before the atomization, mixed and then atomized. This variant is preferably used when the mixtures of two components are stable for only a short time in one liquid phase.
The two liquids are fed separately, atomized separately and mixed before entering the nozzle orifice. This variant can be used for two immiscible solutions or where the stability of the components is ensured only in the solid-state.
It was possible to use these specific types of atomization for the invention just as well as classical two-component atomization.
A process according to the invention is optimal with an inlet air temperature in the range between 100xc2x0 C. and 180xc2x0 C. Since there is also a risk of decomposition of the spray embedding as the inlet air temperature increases, higher temperatures=200xc2x0 C. are normally not productive but can certainly be considered for specific applications. It is surprising that the stability of the biological materials in the formulations is very good although the hot air used according to the invention for the drying preferably has a temperature of  greater than 100xc2x0 C.
The spraying of the solution after the drying results in preferentially spherical particles in an adjustable and reproducible particle size range. Spherical shapes with favourable flow properties and a particular particle size range are precisely those particularly advantageous for many types of pharmaceutical application.
The drying times with the process according to the invention are not more than one minute.
It is also possible according to the invention to produce granules by spraying the solution of biological material and substance mixture onto a carrier with a particle size range from 0.010 mm to 10 mm, preferably 0.1 to 1 mm.
It is thus possible with the process according to the invention, by using the stated substance mixtures, to improve crucially the drying of carbohydrate-containing compositions. The formulations according to the invention contain as main component a carbohydrate, preferably sugar, and one or more zwitterions with polar or apolar radicals or derivatives thereof, with the glass transition temperature of the sugar being distinctly increased by this zwitterion addition.
Formulations in which the main component are zwitterions with polar or apolar radicals or derivatives thereof can be converted into stable amorphous forms by addition of carbohydrates.
It is also possible as an alternative to the carbohydrates to use zwitterions with polar or apolar radicals or derivatives thereof or mixtures thereof. These formulations can be composed of at least 2 zwitterions or derivatives thereof with polar radicals, at least 2 zwitterions or derivatives thereof with apolar radicals or of at least one zwitterion with polar and apolar radical.
Amorphous and dry formulations can also be obtained by specific adjustment of the pH of the solution of zwitterions with polar or apolar radicals or derivatives thereof, alone or in combination.
Substance mixtures which can be preferably employed according to the invention consist of at least two substances selected from sucrose, L-arginine, L-phenylalanine, L-aspartic acid, L-isoleucine and derivatives thereof.
Besides the compositions indicated in the examples, the following substance mixtures have also proved particularly suitable as matrix for the formation of sprayed products according to the invention:
Mixture (formula) 1:
Sucrose, L-arginine and L-phenylalanine,
Mixture (formula) 2:
L-Arginine, L-aspartic acid and L-isoleucine,
Mixture (formula) 3:
L-Arginine and L-phenylalanine,
Mixture (formula) 4:
L-Arginine, L-phenylalanine and L-aspartic acid.
The invention also relates to the use of the amorphous products produced according to the invention for producing diagnostic or therapeutic compositions by further processing them where appropriate with conventional ancillary substances and excipients.